The present invention utilizes fluorescent acidotropic probes for the labeling and tracing of acidic organelles in cells and cell-free systems. Acidotropic probes selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes in cultured animal cells.
While some weakly basic amines have been shown to selectively accumulate in cellular compartments with low internal pH, there have been relatively few acidic organelle stains available for the researcher. The most frequently used acidotropic probe, N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride (hereafter referred to as DAMP), is not fluorescent and requires fixation and permeabilization of the cell, followed by the use of anti-DNP antibodies conjugated to a fluorophore, an enzyme or ferritin in order to visualize the staining pattern. The fluorescent probes neutral red and acridine orange are also commonly used for staining acidic organelles, but they lack specificity and are not well retained in the organelles, particularly after fixing and permeabilization.
The compound dansyl cadaverine has been described as a lysosomotropic reagent (Verhoef, et al. J. IMMUNOLOGY 131, 125 (1983); Vandenbroucke-Grauls, et al. IMMUNOLOGY 51, 319 (1984)), however dansyl cadaverine is only described as having an effect on the function of human natural killer cells and human polymorphonuclear leucocytes. More recent research describes monodansyl cadaverine as a fluorescent label, however it is described as useful as a label for autophagic vacuoles, as it fails to label either endosomal compartments or lysosomes (Biederbick et al. EUR. J. CELL BIOL. 66, 3 (1995). In addition, monodansyl cadaverine possesses several additional disadvantages. The dansyl fluorophore is excited in the ultraviolet region (&lt;350 nm), which is generally incompatible with living systems, has a low quantum yield and has a low extinction coefficient (less than 5,000) requiring high concentrations of dye when staining cells.
The method of the present invention stains acidic organelles rapidly and brightly, and is useful for both short-term and long-term tracking studies. The dyes used in the current method are highly selective for acidic organelles, label living cells at submicromolar concentrations of probe, and exhibit bright fluorescent staining and good photostability. In addition, many of the dyes of the invention are well-retained in acidic organelles, even after aldehyde fixation. The current invention uses a dipyrrometheneboron difluoride fluorophore linked to a weak base that is only partially protonated at neutral pH.
Certain characteristics of dipyrrometheneboron difluoride dyes are known. The fluorescence of simple substituted dipyrrometheneboron difluoride derivatives, comparable to that of fluorescein, was previously noted by Treibs & Kreuzer (Difluorboryl-Komplexe von Di- und Tripyrrylmethenen, LIEBIGS ANN. CHEM. 718, 208 (1968)). Use of such dyes for dye laser systems is described in U.S. Pat. No. 4,916,711 to Boyer, et al. (1990) (incorporated by reference). The use of simple dipyrrometheneborondifluoride dyes as sensitizing agents to enhance photodynamic therapy (PDT) has been specifically described by Boyer (U.S. Pat. No. 5,189,029; incorporated by reference). Chemically reactive dipyrrometheneboron difluoride dyes that can be coupled to other molecules are described in U.S. Pat. No. 4,774,339 to Haugland, et al. (1988) (incorporated by reference). This patent describes dipyrrometheneboron difluoride dyes that possess high quantum yields, insensitivity of fluorescence properties to changes in pH, high absorbance, and a broad range of solubilities. The references, however, do not describe the utility of dipyrrometheneboron difluoride dyes that possess a weakly basic amine moiety for preferentially staining acidic organelles. Haugland et al. (1988) fails to specifically describe dipyrrometheneboron difluoride dyes having substituents that are in turn substituted by tertiary aliphatic amines, primarily as the reference is describing chemically reactive fluorophores, and a tertiary amine is not typically considered to be a chemically reactive group, in the sense of being useful for preparing labeled conjugates.
More recently, additional dipyrrometheneboron difluoride dyes with advantageous long-wavelength characteristics have been identified, including ethenyl-substituted dipyrrometheneboron difluoride dyes as described in U.S. Pat. No. 5,187,288 to Kang et al. (1993) (incorporated by reference); heteroaryl-substituted dipyrrometheneboron difluoride dyes as described in U.S. Pat. No. 5,248,782 to Haugland, et al. (1993) (incorporated by reference); and aza-dipyrrometheneboron difluoride dyes as described in U.S. Pat. No. 5,446,157 to Morgan et al. (1995) (incorporated by reference). In addition, chemically reactive long wavelength dyes have been described in U.S. Pat. No. 5,274,113 to Kang, et al. (1993) (incorporated by reference); and benzo-substituted dipyrrometheneboron difluoride dyes as described in U.S. Pat. No. 5,433,896 to Kang, et al. (1995) (incorporated by reference). While the preparation of amino and aminoalkyl substituted dyes were described in the above patents, none of the patents describe the utility of such compounds particularly for preferentially staining acidic organelles. The above patents that are directed at reactive dyes also fail to describe dyes having substituents that are in turn substituted by tertiary aliphatic amines, as tertiary amines are typically not considered to be chemically reactive groups. Previously, primary aminoalkyl substituents have typically been utilized to provide a site for conjugating the dipyrrometheneboron difluoride dye to a desired biomolecule.
The acidic organelle stains of this invention are freely permeant to membranes of intact cells and can be used for staining cells at very low concentrations of dye that are not toxic to living cells or tissues. The instant dyes and method are useful for investigating the biogenesis of lysosomes, the development of autophagic vacuoles, investigating retina and cultured neurons, and detecting pH gradients. The current invention is also useful for labeling cells that possess acidic organelles, such as yeast, spermatozoa and plant cells.